Strong plastid degradation is consistent within section Chondrophyllae, the most speciose lineage of Gentiana

Abstract Recovering phylogenetic relationships in lineages experiencing intense diversification has always been a persistent challenge in evolutionary studies, including in Gentiana section Chondrophyllae sensu lato (s.l.). Indeed, this subcosmopolitan taxon encompasses more than 180 mostly annual species distributed around the world. We sequenced and assembled 22 new plastomes representing 21 species in section Chondrophyllae s.l. In addition to previously released plastome data, our study includes all main lineages within the section. We reconstructed their phylogenetic relationships based on protein‐coding genes and recombinant DNA (rDNA) cistron sequences, and then investigated plastome structural evolution as well as divergence time. Despite an admittedly humble species cover overall, we recovered a well‐supported phylogenetic tree based on plastome data, and found significant discordance between phylogenetic relationships and taxonomic treatments. Our results show that G. capitata and G. leucomelaena diverged early within the section, which is then further divided into two clades. The divergence time estimation showed that section Chondrophyllae s.l. evolved in the second half of the Oligocene. We found that section Chondrophyllae s.l. had the smallest average plastome size (128 KB) in tribe Gentianeae (Gentianaceae), with frequent gene and sequence losses such as the ndh complex and its flanking regions. In addition, we detected both expansion and contraction of the inverted repeat (IR) regions. Our study suggests that plastome degradation parallels the diversification of this group, and illustrates the strong discordance between phylogenetic relationships and taxonomic treatments, which now need to be carefully revised.


| INTRODUC TI ON
The increasing availability of plastid genomes represents an excellent opportunity to explore phylogenetic relationships and molecular evolution in plants (Twyford & Ness, 2017). For example, plastid phylogenomics permitted the resolution of some persistent taxonomic uncertainties in challenging plant groups (e.g., Lamiaceae; Zhao et al., 2021), and led to a better understanding of evolutionary patterns both in some selected taxa (e.g., evolutionary radiations in Saussurea; Zhang, Landis, et al., 2021) and in major lineages (e.g., Jurassic gap in angiosperms; Li, Yi, et al., 2019).
Furthermore, comparing plastome structure among related clades and linking the structural changes with diversification can offer clues to the mechanisms driving their evolution (Wicke et al., 2016).
In land plants, plastid genomes are generally composed of two inverted repeat (IR) regions that are separated by the large single copy (LSC) region and the small single copy (SSC) region (Jansen & Ruhlman, 2012). Although plastome structure is usually conservative in plants (Mower & Vickrey, 2018), comparative analysis among closely related taxa can provide insights into the evolution of plastid genomes, as for example the expansion/contraction of the IR (Choi et al., 2019;Weng et al., 2017) and gene loss Mower et al., 2021;Yao et al., 2019).
Gentians have long attracted the attention of scientists because of their medical, chemical, and horticultural value (Ho & Liu, 2001;Rybczyński et al., 2015). Gentiana species are predominantly alpine, and their main center of diversity is located in the Qinghai-Tibet Plateau (QTP). This area further acted as the primary source for dispersal to many other distant mountainous regions of the world (Favre et al., 2016;Ho & Liu, 2001). Although Gentiana is subcosmopolitan, only one species-rich section (182 species, 51.7% of all Gentiana), namely section Chondrophyllae Bunge sensu lato (s.l.), is almost globally distributed, whereas another 11 sections of Gentiana are endemic to one or two continents (Favre et al., 2016;Ho & Liu, 2001).
Section Chondrophyllae s.l. is a well-supported monophyletic group that diverged in the first half of the Miocene (Favre et al., 2016;, and includes former sections Chondrophyllae Bunge s. str., Dolichocarpa T. N. Ho, and Fimbricorona T. N. Ho which are intermixed and paraphyletic (Favre et al., 2016. Often based upon minute morphological traits, section Chondrophyllae s. str was divided into 10 series (Ho & Liu, 2001), an example being series Fimbriatae Marquand, which is characterized only by filiform calyx lobes and fringed plicae.
Although our understanding of the taxonomy and phylogenetic relationships among Gentianeae genera and within Gentiana has greatly improved in the past decades (e.g., Favre et al., 2010Favre et al., , 2020, little is known about the phylogenetic relationship and pattern of molecular evolution in section Chondrophyllae s.l. itself, and more specifically among its series. For example, it is unclear whether the intrasectional lineages of section Chondrophyllae s.l. are monophyletic. Furthermore, a karyological study revealed varying basic chromosome numbers in the section without any obvious clustering according to series (Küpfer & Yuan, 1996). The phylogenetic relationships within section Chondrophyllae s.l. were first studied using internal transcribed spacer (ITS) data, resulting in a poorly supported tree (Yuan & Küpfer, 1997). When more DNA fragments were included, the phylogenetic resolution improved, but the intrasectional relationships were still not resolved Favre et al., 2016).
Preliminary plastome data showed a great potential to reconstruct a robust phylogeny for section Chondrophyllae s.l., although a limited number of species was included . In addition, cytonuclear discordance was observed in section Chondrophyllae s.l. , a possible sign of hybridization, thus showing that maternally inherited DNA might be a promising way to trace the evolutionary history of this group. Furthermore, previous studies have showed that section Chondrophyllae s.l. has the most notable plastome size decreases and microstructural changes in the whole subtribe Gentianinae, following gene losses, IR contraction, and SSC reduction . However, these studies did not include all lineages of Chondrophyllae s.l. (4 series out of 10). Genome reduction is believed to parallel high evolutionary rate (Wicke et al., 2016) and evolutionary radiations (Kapusta et al., 2017;Moraes et al., 2022). Therefore, more plastomes are needed to verify whether plastome degradation is an ubiquitous trend in section Chondrophyllae s.l., and whether it relates to the radiation of this group.
In this study, we newly sequenced plastomes of 21 species belonging to section Chondrophyllae s.l., and combined them with existing plastome data in order to reconstruct a robust tree for this group, and assessed whether plastome microstructural changes and current morphology-based taxonomic treatment are consistent with molecular phylogenetic relationship.

| Taxon sampling
A total of 21 species (22 individuals) were sampled representing the 10 main series of section Chondrophyllae s.l. (Table 1; Table S1).
Usually, plants of this section are minute annuals, and thus a whole  (Table S1).

| Sequencing, assembly, and annotation
Total genomic DNA isolation, DNA fragmentation, and sequencing library construction followed the methodology described in Fu et al. (2016). The genomic DNA library of each species was sequenced using the Illumina HiSeq 2500 platform (Novogene), yielding about 2 Gb of 150-bp paired-end reads. The plastome was assembled using GetOrganelle v.1.7.1 (Jin et al., 2020) with the default parameters. Each plastid genome was annotated with GeSeq (Tillich et al., 2017) and PGA (plastid genome annotator) (Qu et al., 2019). All plastome sequences were saved as GB2sequin files (Lehwark & Greiner, 2018) and deposited in GenBank (Table 1). In addition to the 22 newly sequenced plastomes, 7 another plastomes in section Chondrophyllae s.l. were retrieved from GenBank for downstream analysis (Table 1). Moreover, the entire rDNA cistron TA B L E 1 Plastome structure and sequence information for species of Gentiana section Chondrophyllae s.l. included in this study. Note: Newly sequenced plastomes are indicated with asterisks (*) after the GenBank accession numbers. Columns LSC, IR, and SSC report the length of the large single-copy, inverted repeat, and small single-copy regions, respectively, calculated in base pairs.
was also assembled using GetOrganelle v.1.7.1 (Jin et al., 2020) with the default parameters. The rDNA cistron sequences were deposited in GenBank (ON543454-ON543484) and their details are presented in Table S2.

| Phylogenetic analysis
We used the 29 plastomes available in section Chondrophyllae s.l.
to reconstruct phylogenetic relationships among lineages. Twelve plastomes representing several other sections of Gentiana were retrieved from GenBank to server as outgroup ( were removed as burn-in. For rDNA cistron data, ML and BI trees were built following the methodology described above.

| Plastome structural changes
Genome comparisons were conducted to identify structural differences using mVISTA (Frazer et al., 2004). The genes on the boundaries of the junction sites of the plastome were visualized in IRscope (Amiryousefi et al., 2018). We tested whether plastome size changes have phylogenetic signal using Pagel's lambda (Pagel, 1997(Pagel, , 1999 in the R package MOTMOT (Puttick et al., 2020). G. faucipilosa and G. shaanxiensis were not included in the phylogenetic signal analysis due to their incomplete plastomes in this study.

| Divergence dating
Using the protein-coding matrix, the divergence times of main lineages were estimated using the Bayesian method implemented in BEAST v.2.4 (Bouckaert et al., 2014;Drummond et al., 2012). We ran the analyses using the Hasegawa-Kishono-Yano (HKY) substitution model, the Yule model, and strict clock model. To improve the accuracy of the molecular dating, we constrained two nodes strictly following the settings in . The stem node of G.
sect. Cruciata was constrained with a fossil from the early Miocene (Mai, 2000), We ran three independent MCMC with 10 million generations, sampling every 1000th generation and discarding the initial 20% as burn-in. Convergence was judged as suitable by ESS values (>200).

| Phylogenetic relationship and divergence time
After filtering, the phylogenetic data matrix included 58 proteincoding genes shared among all samples. The matrix resulted in a strongly supported topology of section Chondrophyllae s.l.
After G. intricata was removed, the support of the uncertain node was improved (BS = 77%, PP = 1.0; Figure S1). We found that G. capitata and G. leucomelaena were early diverged within section Chondrophyllae s.l., which was then further divided into two main clades.  (Figure 1). This is, for example, the case for species of series Humiles, which are distributed throughout the tree (Figure 1).
A total of 31 rDNA cistron from 28 species (including the outgroup) were assembled in this study (Table S2)

| Plastome microstructural changes
When compared to other closely related sections (e.g., section Cruciata), we found that section Chondrophyllae s.l. had a similar plastome structure overall. Furthermore, one gene complex (ndh) and rps16, along with their respective flanking regions, were fully or partly lost in the entire section Chondrophyllae s.l., and three introns (rpoC1 intron, rpl2 intron, and clpP 2nd intron) have been lost in some samples ( Figure S3). An expansion of the IR was observed in G. loureirii and G. grata. In G. loureirii, the expansion was caused by the transfer of three plastid genes (ycf1, rps15, and partial ndhH) from the SSC to the IR region (Figure 4). In G. grata, the F I G U R E 1 Phylogenetic tree and variation of plastid size in Gentiana section Chondrophyllae sensu lato. The topology is derived from an analysis of 58 plastid protein-coding genes. Phylogenetic support values for both maximum likelihood (ML) and Bayesian inference (BI) are shown above branches only when they differ from 100% bootstrap support (BS) and 1.00 posterior probability (PP). Heatmaps illustrate changes in plastid size (LSC, IR, SSC, and total) with relatively reduced sizes in blue and relatively larger sizes in red. The taxonomic attribution of each sample is indicated by colored square with black frame.
IR expansion was due to the transfer of ycf1 from the SSC to the IR region. We also observed a contraction of the IR in G. spathulifolia due to the transfer of genes rrn5,and rrn4.5) from the IR to the SSC region ( Figure 4). Finally, the contraction of SSC was common in the entire section Chondrophyllae s.l., and was due to substantial sequence loss (e.g., ndh complex, Figure 4;

| Phylogenetic relationships, taxonomic treatments, and possible reticulate evolution
Recovering the phylogenetic relationships of intensively diversifying taxa has always been a challenging task in evolutionary studies (Olave & Meyer, 2020;Thomas et al., 2021). Using plastome data, we recovered a well-supported phylogenetic tree and resolved the relationship among the species included in this study with a much improved resolution in comparison to previous molecular studies on F I G U R E 2 Phylogenetic tree of Gentiana section Chondrophyllae sensu lato based on recombinant DNA (rDNA) cistron sequences. Numbers on the branches represent bootstrap supports in maximum-likelihood (ML) analyses and posterior probabilities (PP) in Bayesian inference (BI) analysis. Taxonomic attribution of each sample is indicated by colored square.
section Chondrophyllae Favre et al., 2016;Yuan & Küpfer, 1997). The phylogenetic power of our study, harnessed from genomic data, thus echoes that reported in an increasing number of similar investigations on the evolutionary history of radiating alpine taxa, such as Rhodiola (Zhao et al., 2020) and Saussurea (Zhang, Landis, et al., 2021;Zhang, Yu, et al., 2021).
Furthermore, we found that the currently recognized taxonomic treatment within section Chondrophyllae s.l. (e.g., Ho & Liu, 1990) is relatively inconsistent with phylogenetic relationship we recovered in the trees based on plastome and rDNA cistron sequences (Figures 1 and 2). For example, the two better-sampled groups in our study, namely series Dolichocarpa and Humiles, were not monophyletic (Figure 1). Although the number of Chondrophyllae s.l. species included in this study is limited (26 out of ca. 180), we believe increasing the number of samples would not recover monophyletic clades for series Dolichocarpa and Humiles, given that all other known main lineages within the section were included. Also, the results of other studies showed the same pattern , including with Sanger sequencing with a much higher proportion of species (Favre et al., 2016). Reticulate evolution is likely to be a major contributor to this inconsistent pattern, as well as to an accelerated diversification. Reticulate evolution was also suggested in Swertia, another species-rich genus of Gentianeae in the Tibeto-Himalayan region (Chassot et al., 2001), as well as in other taxa such as woody bamboo , Lachemilla (Morales-Briones et al., 2018), and even lizards (Esquerré et al., 2022). Hybridization is at the source of reticulate evolution, and in Gentiana, interspecific crosses were detected in several sections in both the region of the Qinghai-Tibet Plateau (QTP) and Europe Hu et al., 2016). However, no direct evidence of hybridization was ever reported in section Chondrophyllae s.l., although cytonuclear discordances in the phylogeny produced in this study, as well as other evidence based upon transcriptome data  suggest that hybridization could be common also in this group. In fact, current and past hybridization events are only poorly investigated as potential contributor to diversification in the alpine biome of the region of the QTP. This shortcoming is for example most visible in Saxifraga, for which it was reported that hybridization was intense in Europe and almost absent in the region of the QTP (Ebersbach et al., 2020). In summary, evidence suggests that the current taxonomic treatment within section Chondrophyllae s.l. needs to be revised with the help of advanced molecular data and an increased species cover, and that the extent of past and present events of hybridization should be evaluated in this section.

| Is plastome degradation related to radiation?
Plastome degradation is visible as the loss of genes and sequences, and was observed in a wide range of vascular plant lineages (Lehtonen & Cárdenas, 2019;Mohanta et al., 2020;Yao et al., 2019). Having sampled all main morphological lineages of section Chondrophyllae s.l., our study identified a strong and consistent plastome degradation in this group. Indeed, section Chondrophyllae s.l. displays the shortest average plastome sizes (128 Kb) in Gentianeae, as sister subtribes Gentianinae and Swertiinae were found to have plastome sizes ranging from 135 to 151 Kb  and from 149 to 153 Kb (Zhang, Sun, et al., 2020;Zhang, Yu, et al., 2021), respectively. Shorter plastomes in this case are due to structural changes such as SSC contraction and frequent gene losses, but did section Chondrophyllae s.l. experience rapid diversification or even explosive radiation?
First, we need to keep in mind that species of section Chondrophyllae s.l. are usually characterized by long branches in phylogenetic trees (Figure 1; e.g., . Hence, this clade has accumulated many more genetic modifications than closely related lineages in the same lapse of time, suggesting a higher molecular evolution than other sections in Gentiana. This is indirectly supported by the sheer number of Chondrophyllae species (representing 51.7% of all species in the genus, i.e., 182 species ;Ho & Liu, 2001;Favre et al., 2020), and by a reported accelerated substitution rate, admittedly using a limited sampling .
Second, it was reported that accelerated substitution rates may be associated with plastome size (Schwarz et al., 2017) and life history (e.g., annual vs. perennial; Gaut et al., 2011), and in fact, most species of section Chondrophyllae s.l. are annual, with the exception of series Napuliferae. This series is one of the two species-poor series in the section, containing only three species (Ho & Liu, 2001), and interestingly, series Napuliferae has also the longest plastome (G. loureirii, 138 Kb) in section Chondrophyllae s.l. (based upon currently available data). Thus, it seems that plastome degradation may be correlated with the life cycle and diversification rates in section Chondrophyllae s.l., as suggested by  and observed in other taxa such as Orchidaceae Li, Yi, et al., 2019;Tang et al., 2021). Nevertheless, because some plastome degradation was also observed in a few perennial lineages of Gentianinae Sun et al., 2018) and other perennial plant lineages (e.g., Tang et al., 2021;Zhou et al., 2022), more species of section Chondrophyllae s.l. need to be investigated to understand fully whether this lineage has undergone explosive radiation. In any case, the diversification of section Chondrophyllae s.l. may have been fostered by the climatically and geologically dynamic context of the region of the QTP. As stated by the "Mountain-Geobiodiversity Hypothesis" (Mosbrugger et al., 2018), a species-pump effect is likely to have been a powerful driver of diversification in this region. Indeed, it would be expected that such climate-driven cycles of range expansions and contractions, alternatively forcing allopatry and secondary contacts among closely related (and possibly interfertile) taxa, may have disproportionately affected the diversification of annuals in comparison to perennials. This, however, remains yet to be tested in section Chondrophyllae s.l. and across multiple taxa.

| Conclusion
By sampling the main evolutionary lineages in Gentiana section Chondrophyllae s.l., we have discovered a consistent plastid degradation in the entire clade, including the loss of functional genes and sometimes short single-copy regions. Whether or not section Chondrophyllae s.l. experienced explosive radiation is still partially up for debate, although several lines of evidence (including short plastomes) indicate that it might be the case. A taxonomic revision will be necessary to further understand the mechanisms involved in the evolutionary history of section Chondrophyllae s.l., including hybridization within a context of rapidly changing geological and climatic settings during the last few million years.

ACK N OWLED G M ENTS
We thank Zhi-Zhong Li and Bin Zheng of the Wuhan Botanical Garden,  (31600296) to P.C.F, as well as by the German Science Foundation (Deutsche Forschungsgemeinschaft, FA1117/1-2) to A.F.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are provided within the text, tables, figures and supplements.